The present invention relates to the use of a nucleotide sequence derived from the 5xe2x80x2 end of the genomic RNA or of the proviral DNA of a reticuloendotheliosis virus as internal ribosome entry site (IRES) and/or for improving retroviral encapsidation. More particularly, it relates to expression vectors comprising this sequence and in particular polycistronic vectors allowing the effective and stable expression of several genes of interest under the control of the same promoter. The present invention finds an advantageous application in the field of vectors for gene therapy.
The feasibility of gene therapy applied to humans no longer needs to be demonstrated, and this relates to numerous therapeutic applications such as genetic diseases, infectious diseases and cancers. Numerous prior art documents describe the means using gene therapy, in particular by means of viral vectors. The vectors are generally obtained by deletion of at least part of the viral genes which are replaced by the therapeutic genes of interest. Such vectors can be propagated in a complementation line which provides in trans the viral functions deleted in order to generate a viral particle defective for replication but capable of infecting a host cell. To date, retroviral vectors are among the most widely used but there may also be mentioned vectors derived from adenoviruses, adeno-associated viruses, pox viruses and herpes viruses. This type of vectors, their organization and their mode of infection are widely described in the literature accessible to persons skilled in the art.
As a guide, the retroviral genome consists of a single-stranded linear RNA of positive polarity. In addition to the regulatory sequences R and U5 and U3 and R present at the 5xe2x80x2 and 3xe2x80x2 ends respectively, it carries three genes: gag encoding the capsid proteins, pol encoding reverse transcriptase and integrase and env encoding the envelope proteins. The encapsidation signals, situated upstream of the U5 sequences up to the beginning of the coding region of the gag gene, participate in the dimerization and the encapsidation of the viral RNA into the viral particles. The 5xe2x80x2 end of the genome comprises a cap and the 3xe2x80x2 end is polyadenylated. During the infectious cycle, the viral RNA is converted to a double-stranded linear proviral DNA provided at each end with inverted repeat sequences LTR (for Long Terminal Repeat) which are necessary for the initiation of transcription. The latter, which is carried out by the cellular machinery, allows the production of genomic and subgenomic RNAs from which the viral proteins are synthesized. Retroviruses may be classified into 4 subgroups A to D, on the basis of their morphology. Type C groups together the majority of retroviruses including the MLV (Murine Leukemia Virus) and MSV (Murine Sarcoma Virus) viruses used in most of the gene therapy vectors and the REV viruses (Reticuloendotheliosis Virus) from which the nucleotide sequence of the present invention is derived.
It may be advantageous to have gene therapy vectors which are more effective and capable in particular of efficiently producing several proteins of interest. However, the presence of several promoters within the same vector very often results in a reduction or even in a loss of expression over time. This is due to a well known phenomenon of interference between the promoter sequences. In this context, the publication of international application WO 93/03143 provides a solution to this problem which consists in using an internal ribosome entry site (IRES). It describes a dicistronic retroviral vector for the expression of two genes of interest placed under the control of the same promoter. The presence of a picornavirus IRES site between them allows the production of the product of expression derived from the second gene of interest by internal initiation of the translation of the dicistronic mRNA.
Normally, the entry of the ribosomes at the level of the messenger RNA (mRNA) occurs via the cap situated at the 5xe2x80x2 end of all eukaryotic mRNAs. The 40S ribosomal subunits move along the RNA until an appropriate AUG codon is encountered in order to start the protein synthesis. Generally, the initiation takes place at the level of the first AUG codon. However, if the latter is in a context which is not very favorable, the 40S subunits continue up to a subsequent AUG codon situated in a better translational context (Kozak, 1984, Nucleic Acid Res. 12, 3873-3893; Kozak, 1991, J. Biol. Chem. 266, 19867-19870; Pain, 1996, Eur. J. Biochem. 236, 747-771).
However, there are exceptions to this universal rule. The absence of a cap in certain viral mRNAs suggests the existence of alternative structures allowing the entry of the ribosomes at an internal site of these RNAs. To date, a number of these structures, called IRESs because of their function, have been identified in the noncoding 5xe2x80x2 region of noncapped viral mRNAs such as that in particular of the picornaviruses such as the poliomyelitis virus (Pelletier et al., 1988, Mol. Cell. Biol. 8, 1103-1112) and EMCV (Encephalomyocarditis virus (Jang et al., 1988, J. Virol. 62, 2636-2643). Cellular mRNAs possessing IRES elements have also been described. There may be mentioned those encoding the BIP protein (for Immunoglobulin heavy chain binding protein; Macejak and Sarnow, 1991, Nature 353, 90-94), certain growth factors (Teerink et al., 1995, Biochem. Biophy. Acts 1264, 403-408; Vagner et al., 1995, Mol. Cell. Biol. 15, 35-44), the translational initiation factor eIF4G (Gan and Rhoads, 1996, J. Biol. Chem. 271, 623-626) and two yeast transcriptional factors TFIID and HAP4 (Iizuka et al., 1994, Mol. Cell. Biol., 14, 7322-7330). IRES sites have also been demonstrated in the VL30-type murine retrotransposons (Berlioz et al., 1995, J. Virol. 69, 6400-6407) and, more recently in the mRNAs encoding the gag precursor of the Friend (FMLV) and Moloney (MoMLV) murine leukemia viruses (Berlioz and Darlix, 1995, J. Virol. 69, 2214-2222; Vagner et al., 1995, J. Biol. Chem. 270, 20376-20383).
A new internal ribosome entry site has now been found in the noncoding 5xe2x80x2 region of the RNA of the avian reticuloendotheliosis virus (REV) type A (REV-A) and its efficiency for initiating the translation of coding sequences placed after it in a monocistronic or dicistronic manner has been shown.
The IRES site of the present invention is particularly advantageous compared with those already described in the literature. In the first place, it allows a high level of expression of the cistron which it controls. In addition and, unexpectedly, it can also, within the framework of a retroviral vector, contribute or improve, in association with an appropriate encapsidation region, the dimerization or encapsidation functions, allowing an increase in the viral titer. And finally, because of its weak homology with the murine retrovirus sequences used in most of the gene therapy vectors intended for human use, its use considerably reduces the risk of production of replication-competent viruses.
Most of the gene therapy protocols approved by the RAC (Recombinant DNA Advisory Committee) in the United States use vectors derived from the MoMLV virus. Currently, the choice of a specific retroviral vector for a given therapeutic application remains empirical and the factors influencing the viral titer and the expression of the genes have not yet been clearly elucidated. The study of the cis-acting sequences which control the encapsidation and the establishment of the relative strengths of the various IRES elements can make it possible to optimize the gene therapy vectors in terms of titer and gene expression. One of the aims of the present invention is to provide new retroviral vectors capable of being propagated at a high titer and of allowing optimal expression of one or more genes of interest.
Accordingly, the subject of the present invention is the use of a nucleotide sequence derived from all or part of the 5xe2x80x2 end of the genomic RNA of a type C retrovirus with the exception of the Friend (FMLV) and Moloney (MoMLV) murine leukemia viruses, as internal ribosome entry site (IRES) in a vector and/or for allowing or improving the encapsidation of a retroviral vector.
Nucleotide sequence is understood to mean a sequence composed of ribo-(RNA) or deoxyribonucleotides (DNA). Within the framework of the present invention, the 5xe2x80x2 end of the genomic RNA of a retrovirus corresponds to the 5xe2x80x2 quarter of said RNA which extends from the site of initiation of transcription (nucleotide +1) to about 2 kb in the 3xe2x80x2 direction. The term retrovirus is widely defined in basic virology manuals accessible to persons skilled in the art and the essential characteristics have been summarized as a guide above. The term xe2x80x9cderivedxe2x80x9d refers to a sequence having a type C retroviral origin, but which may have undergone at least one modification in relation to the native sequence. The modification(s) which may be envisaged include the deletion, addition, substitution and/or mutation of one or more nucleotides (nt). Such modifications may be designed, for example, to increase the IRES function, the encapsidation function or the function of introducing suitable restriction sites in order to facilitate subsequent cloning steps. The term xe2x80x9cderivativexe2x80x9d also comprises the DNA equivalent of the genomic RNA in a modified or unmodified form.
IRES denotes a site capable of promoting the entry of the ribosomes into an RNA molecule in a manner independent of the cap. In accordance with the aims pursued by the present invention, the IRES function can be exerted in any expression cassette or vector. A sequence in use within the framework of the present invention may also act as element activating the encapsidation of retroviruses or retroviral vectors by promoting the dimerization of two copies of the retroviral genome and/or the encapsidation of the dimer into the viral particles. According to a preferred embodiment, said sequence is capable of exerting an IRES function and of improving the encapsidation function when it is introduced into an appropriate retroviral vector.
A nucleotide sequence as used within the framework of the present invention may be isolated from the 5xe2x80x2 end of the genomic RNA or of the proviral DNA of a type C retrovirus or of any state of the art plasmid carrying the retroviral fragment of interest. It goes without saying that it can be generated by any technique used in the art, for example by cloning with the aid of appropriate probes, by PCR (Polymerase Chain Reaction) or alternatively by chemical synthesis. Advantageously, said sequence comprises all or part of the region which follows the U3 domain of the 5xe2x80x2 LTR, up to the initiator AUG codon of the gag gene. For the purposes of the present invention, it comprises at least 50 nucleotides, advantageously at least 100 nucleotides, preferably at least 200 nucleotides and preferably at least 300 nucleotides included in said 5xe2x80x2 end. However, it can of course extend beyond in the 5xe2x80x2 or 3xe2x80x2 direction or comprise additional sequences. Advantageously, said sequence comprises from 100 to 1500 nucleotides and, in particular, from 300 to 800 nucleotides.
It is preferable to use within the framework of the present invention a type C retrovirus with the exception of the FMLV and MoMLV viruses. A type C retrovirus which is more particularly suitable is selected from the REV (reticuloendotheliosis virus) and MSV (murine sarcoma virus) viruses and in particular the Moloney (MMSV), MHV (Mus hortulanus virus), MEV (mouse endogenous retrovirus), FMOV (FBR murine osteosarcoma virus), AMLV (AKV murine leukemia virus), MEELV (mouse endogenous ecotropic murine leukemia virus), SFFV (Friend spleen focus-forming virus), RASV (rat sarcoma virus), FLV (Feline leukemia virus), FSV (feline sarcoma virus), EFLV (cat endogenous proviral feline leukemia virus), SSV (Simian sarcoma virus), GALV (gibbon ape leukemia virus) and BAEV (baboon endogenous virus) viruses.
According to a most preferred embodiment, a nucleotide sequence used in the present invention is derived from all or part of the 5xe2x80x2 end of the genomic RNA of a reticuloendotheliosis virus (REV). The REV viruses comprise in particular various A, B and T sub-types as well as the DIAV (duck infectious anemia virus), SNV (spleen necrosis virus) and CSV (chick syncytial virus) viruses (see for example Encyclopedia of Virology, 1994, Enrietto, Reticuloendotheliosis viruses, p. 1227-1232 Ed. R. Webster and A. Granoff, Academic Press, Hartourt Brace xc2xa7 Company Publishers). An REV virus which is most particularly suitable is the avian reticuloendotheliosis virus, in particular the type A virus (REV-A).
According to the latter variant, a nucleotide sequence comprising at least 100 nucleotides and at most 800 nucleotides (nt) of the noncoding SI end of the REV-A virus and more particularly a nucleotide sequence which is substantially homologous or identical to all or part of the sequence presented in the sequence identifier SEQ ID NO: 1 will be preferably used. As preferred examples, there may be mentioned a nucleotide sequence which is substantially homologous or identical to the sequence presented in the sequence identifier SEQ ID NO: 2:
(i) starting at nucleotide 1 and ending at nucleotide 578,
(ii) starting at nucleotide 265 and ending at nucleotide 578, or
(iii) starting at nucleotide 452 and ending at nucleotide 578.
The term substantially homologous refers to a degree of homology greater than 70%, advantageously greater than 80%, preferably greater than 90% and, most preferably, greater than 95%. As already indicated, said nucleotide sequence may have a sequence which is slightly different from that described in SEQ ID NO: 1 or 2, provided, however, that the modification(s) does (do) not affect its IRES and/or encapsidation functions.
According to an advantageous mode, the nucleotide sequence used within the framework of the present invention is identical to the sequence presented in the sequence identifier SEQ ID NO: 2:
(i) starting at nucleotide 1 and ending at nucleotide 578,
(ii) starting at nucleotide 265 and ending at nucleotide 578, or
(iii) starting at nucleotide 452 and ending at nucleotide 578.
The IRES function of said nucleotide sequence is particularly advantageous in a context low in magnesium ion, for example in a cellular context. A high concentration of Mg2+ ions may reduce the efficiency of the initiation of translation mediated by the sequence.
A nucleotide sequence used in the present invention is more particularly intended to be integrated into a vector for the transfer and expression of one or more genes of interest. The choice of such a vector is wide and the techniques for cloning into the vector chosen are within the capability of persons skilled in the art. In accordance with the aims pursued by the present invention, it is possible to envisage a plasmid vector or a vector derived from an animal virus and, in particular, from a poxvirus (canarypox or vaccinia virus, in particular Copenhage or MVA), adenovirus, baculovirus, herpesvirus, adeno-associated virus or retrovirus. Such vectors are widely described in the literature. In particular, when an adenoviral vector is used, it may be derived from a human adenovirus (preferably type 2 or 5), an animal adenovirus (preferably canine or bovine) or alternatively from a hybrid between a variety of species. The general technology relating to adenoviruses is disclosed in Graham and Prevec (1991, Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols; Ed. E. J. Murray, the Human Press Inc., p. 109-118).
In accordance with the aims pursued within the framework of the present invention, said nucleotide sequence is preferably positioned upstream of a gene of interest in order to enhance the translation of the product of expression for which it codes. It may be used in an expression cassette of the monocistronic type (for the expression of a gene of interest placed under the control of a promoter) or polycistronic type (for the expression of at least two genes of interest placed under the control of the same promoter). The latter may contain several elements in tandem xe2x80x9cIRES site-gene of interestxe2x80x9d in which at least one of the IRES sites consists of a nucleotide sequence as defined above. The use in a dicistronic cassette, either upstream of the first gene of interest or upstream of the second, is most particularly preferred, the latter variant being preferred.
When a vector according to the invention comprises several expression cassettes, they may be inserted in any orientation relative to each other, either in the same orientation (promoter acting in the same direction) or in reverse orientation (promoter acting in an opposite orientation). Moreover, a vector according to the invention may comprise several nucleotide sequences used according to the invention. In this case, it is preferable that they are derived from different type C retroviruses.
According to a most preferred embodiment, a vector according to the invention is derived from a retrovirus. There may be mentioned, by way of examples, the avian retroviruses such as the avian erythroblastosis virus (AEV), the avian leukemia virus (AVL), the avian sarcoma virus (ASV), the spleen necrosis virus (SNV) and the Rous sarcoma virus (RSV), the bovine retroviruses, the feline retroviruses (FLV, FSV and the like), the murine retroviruses such as the murine leukemia virus (MuLV), the Friend virus (FMLV) and the murine sarcoma virus (MSV) and the primate retroviruses (GALV, FSV, BAEV and the like). Of course, other retroviruses may be used. However, the use of the MoMLV virus is most particularly preferred. The numerous retrovirus vectors described in the literature can be used within the framework of the present invention.
The retroviral vectors which may be envisaged for the purposes of the present invention comprise at least the following elements associated in a functional manner: a retroviral 5xe2x80x2 LTR and a retroviral 3xe2x80x2 LTR, one or more genes of interest, and the nucleotide sequence used within the framework of the present invention to allow or improve the encapsidation of said vector into a viral particle and/or as an IRES site to allow or promote the expression of a gene of interest positioned downstream of said nucleotide sequence. It goes without saying that the retroviral 5xe2x80x2 LTR may be used as a promoter, but it is also possible to use an internal promoter. Moreover, the 5xe2x80x2 and possibly 3xe2x80x2 LTR may have the same retroviral origin (for example REV) as the nucleotide sequence, or a different origin. For example, a monocistronic vector will comprise, from 5xe2x80x2 to 3xe2x80x2, a 5xe2x80x2 LTR, the nucleotide sequence, a gene of interest and a 3xe2x80x2 LTR.
Of course, a retroviral vector according to the invention may also comprise a conventional encapsidation region (E+). However, the presence of the latter is not required when the nucleotide sequence used in the present invention can itself exert the encapsidation function. Such an embodiment may be more particularly envisaged when the retroviral 5xe2x80x2 LTR is derived from an REV virus and, preferably from SNV, and the nucleotide sequence is substantially homologous or identical to the sequence presented in SEQ ID NO: 2, starting at nt 1 and ending at nt 578 or starting at nt 265 and ending at nt 578.
According to an advantageous embodiment, a retroviral vector according to the invention comprises at least:
(a) a retroviral 5xe2x80x2 LTR,
(b) an encapsidation region,
(c) optionally, a first gene of interest followed by an internal promoter region of different origin from that of said retroviral 5xe2x80x2 LTR,
(d) a second gene of interest,
(e) an IRES site,
(f) a third gene of interest, and
(g) a retroviral 3xe2x80x2 LTR,
at least one of the encapsidation region and the IRES site consisting of said nucleotide sequence used according to the invention.
In the case where the retroviral vector according to the invention comprises an expression cassette directed by an internal promoter region, it is preferable, in order to promote gene expression, that the latter is in an opposite orientation relative to the retroviral 5xe2x80x2 and 3xe2x80x2 LTRs. It is also possible to include other elements, for example another IRES site and another gene of interest or another expression cassette.
A preferred retroviral vector according to the invention comprises an encapsidation region which is derived from a murine retrovirus, especially from an MoMLV, or from a VL30-type retrotransposon and an IRES site comprising a nucleotide sequence which is substantially homologous or identical to the sequence presented in the sequence identifier SEQ ID NO: 2:
(i) starting at nucleotide 1 and ending at nucleotide 578,
(ii) starting at nucleotide 265 and ending at nucleotide 578, or
(iii) starting at nucleotide 452 and ending at nucleotide 578.
There may be mentioned in particular the pREV HW-3 and HW-6 type dicistronic retroviral vectors in which the encapsidation region is derived from an MoMLV and the IRES site consists of a nucleotide sequence identical to the sequence presented in SEQ ID NO: 2 starting at nucleotide 265 and ending at nucleotide 578 or starting at nucleotide 452 and ending at nucleotide 578. Of course, persons skilled in the art can vary the genes of interest according to the desired therapeutic effect.
For the purposes of the present invention, a gene of interest used in the invention may be obtained from a eukaryotic organism, prokaryotic organism or a virus by any conventional molecular biological technique. It can encode a polypeptide corresponding to a native protein as found in nature, homologous to the host cell or otherwise, a protein fragment, a chimeric protein obtained from the fusion of polypeptides of various origins or a mutant having improved and/or modified biological properties. Such a mutant may be generated by substitution, deletion and/or addition of one or more amino acid residues. In addition, the polypeptide may be (i) intracellular (ii) membranous, present at the surface of the host cell or alternatively (iii) secreted outside the host cell and may therefore comprise appropriate additional elements, such as a sequence encoding a secretory signal or a region for transmembrane anchorage.
The use of a therapeutic gene of interest encoding a product of expression capable of inhibiting or retarding the establishment and/or the development of a genetic or acquired disease is most particularly preferred. A vector according to the invention is particularly intended for the prevention or treatment of cystic fibrosis, hemophilia A or B, Duchenne""s or Becker""s myopathy, cancer, AIDS, cardiovascular diseases (restenosis, arteriosclerosis, ischemia and the like) and other infectious diseases due to a pathogenic organism: virus, bacterium, parasite or prion. The genes of interest which can be used in the present invention are those which encode the following proteins:
a cytokine and especially an interleukin (IL-2, IL-7, IL-10, IL-12 and the like), an interferon, a tissue necrosis factor and a growth, and especially hematopoietic, factor (G-CSF, GM-CSF),
a factor or cofactor involved in coagulation and especially factor VIII, factor IX, von Willebrand""s factor, antithrombin III, protein C, thrombin and hirudin,
an enzyme and especially trypsin, a ribonuclease, alkaline phosphatase (plap) and xcex2-galactosidase,
an enzyme inhibitor such as xcex11-antitrypsin and viral protease inhibitors
a product of expression of a suicide gene such as thymidine kinase of the HSV virus (herpesvirus) type I, that of the fur1 and/or fcy1 gene of Saccharomyces cerevisiae, ricin,
an activator or an inhibitor of ion channels,
a protein whose absence, modification or the deregulation of whose expression is responsible for a genetic disease, such as the CFTR protein, dystrophin or minidystrophin, insulin, ADA (adenosine diaminose), gluco-cerebrosidase and phenylhydroxylase,
a protein capable of inhibiting the initiation or the progression of cancer, such as the products of expression of the tumor suppressor genes, for example the p53, p73 and Rb genes,
a protein capable of stimulating an immune response, an antibody, the antigens of the major histocompatibility complex or an immunotoxin,
a protein capable of inhibiting a viral infection or its development, for example the antigenic epitopes of the virus in question or altered variants of viral proteins capable of entering into competition with the native viral proteins,
a cellular or nuclear receptor or one of their ligand,
a growth factor (FGF for Fibroblast Growth Factor, VEGF for Vascular Endothelial cell Growth Factor and the like), and
an inducer of apoptosis (Bax and the like), an inhibitor of apoptosis (Bcl2, BclX and the like), a cytostatic agent (p21, p16, Rb and the like), a nitric oxide synthase (NOS), an apolipoprotein (apoAI, apoE and the like), a catalase, an SOD, a factor acting on angiogenesis (PAI for Plasminogen Activator Inhibitor and the like).
Moreover, a gene of interest used in the present invention may also encode a selectable marker which makes it possible to select or identify the host cells transfected with a vector according to the invention. There may be mentioned the neo (neomycin) gene which confers resistance to the antibiotic G418, the dhfr (dihydrofolate reductase) gene, the CAT (Chloramphenicol Acetyl Transferase) gene or alternatively the gpt (xanthine phosphoribosyl) gene.
In general, a promoter which is functional in the host cell considered and, preferably, a human cell will be used for the expression of one or more genes of interest. The choice of the promoter is very broad and within the capability of persons skilled in the art. It may be a promoter which naturally controls the expression of a gene of interest used in the present invention or any other promoter of any origin. Moreover, it may be of a constitutive nature or of a regulatable nature, especially in response to certain tissue-specific or events-specific cellular signals. For example, it may be advantageous to target the expression of the gene of interest at the level of the lymphocytic cells in the case of AIDS, of pulmonary cells in the case of cystic fibrosis or of muscle cells in the case of myopathies.
By way of examples, the promoters which are suitable within the framework of the present invention may be chosen from the SV40 (Simian Virus 40), CMV (Cytomegalovirus), HMG (Hydroxymethyl-Glutaryl Coenzyme A) and TK (Thymidine kinase) promoters, the retroviral LTRs such as that of the MoMLV, RSV or MSV when a retroviral vector is used, the adenoviral promoters E1A and late MLP (Major Late Promoter) especially in the context of an adenoviral vector, the 7.5K, H5R, pK1L, p28 and p11 promoters intended for poxvirus vectors such as the vaccinia virus, the PGK promoter (Phosphoglycerokinase), the liver-specific promoters of the genes encoding xcex11-antitrypsin, factor IX, albumin and transferrin, the promoters of the immunoglobulin genes which allow expression in the lymphocytes, and finally the promoters of the genes encoding the surfactant or the CFTR protein which exhibit a degree of specificity for the pulmonary tissues. They may also be a promoter which stimulates expression in a tumor or cancer cell. There may be mentioned in particular the promoters of the MUC-1 gene which is overexpressed in breast and prostate cancers (Chen et al., 1995, J. Clin. Invest. 96, 2775-2782), CEA (for carcinoma embryonic antigen) gene which is overexpressed in colon cancers (Schrewe et al., 1990, Mol. Cell. Biol. 10, 2738-2748), tyrosinase gene which is overexpressed in melanomas (Vile et al., 1993, Cancer Res. 53, 3860-3864), ERB-2 gene which is overexpressed in cancers of the breast and the pancreas (Harris et al., 1994, Gene Therapy 1, 170-175), xcex1-fetoprotein which is overexpressed in liver cancers (Kanai et al., 1997, Cancer Res. 57, 461-465), APC which is overexpressed in colorectal cancers, BRCA-1 and 2 (Wooster et al., 1995, Nature 378, 789-792) which are overexpressed in ovarian cancers and PSA (for prostate specific antigen) which is overexpressed in prostate cancers.
Moreover, the gene of interest used in the present invention may comprise other sequences which improve its expression, both at the level of transcription and of translation; for example, an enhancer-type transcriptional activator sequence, an intron sequence, a transcriptional termination signal (polyA) and, as indicated above, a secretory signal or a transmembrane region.
The invention also covers the viral particles generated from a viral vector according to the invention. The procedure is generally carried out by transfecting the latter into an appropriate cell line. If the viral vector used is replication-defective, a complementation line will be used. In general, persons skilled in the art know the lines which can be used to generate infectious viral particles as well as the method to be used depending on the vector used.
For example, in the case of an adenoviral vector, the 293 line may be used (Graham et al., 1977, J. Gen. Virol., 36, 59-72). As regards a retroviral vector, the use of ecotropic cell lines, such as the CRE line (Danos and Mulligan, 1988, Proc. Natl. Acad. Sci. USA, 85, 6460-6464) or GP+E-86 line (Markowitz et al., 1988, J. Virol., 62, 1120-1124) may be envisaged. However, the use of an amphotropic complementation line such as the PG13 line (Miller et al., 1991, J. Virol., 65, 2220-2224) or Psi Env-am line (Markowitz et al., 1988, T.A.A.P. Vol. CI, 212-218) is most particularly preferred. Generally, the infectious viral particles are recovered in the culture supernatant for the transfected complementation cells.
The invention also extends to the cells comprising a vector according to the invention or infected with infectious viral particles according to the invention. The methods of transfection are well known to persons skilled in the art. There may be mentioned the technique of precipitation with calcium phosphate, that with DEAE-dextran, microinjection or encapsulation into lipid vehicles. Moreover, the vectors according to the invention may be present in the host cell in a form integrated into the cellular genome or in the form of episomes both in the nucleus and in the cytoplasm. The cell according to the invention is advantageously a eukaryotic cell, especially a mammalian cell and, preferably, a human cell. It may be a primary or tumor cell of hematopoietic origin (totipotent stem cell, leucocyte, lymphocyte, monocyte, macrophage and the like), hepatic origin, epithelial origin, fibroblast, from the central nervous system and, most particularly, a muscle cell (myoblast, myocyte, satellite cell, smooth muscle cell and the like), cardiac cell, vascular cell, trachea cell, pulmonary cell or cell from the central nervous system.
The present invention also relates to the therapeutic use of a vector, of a viral particle or of a cell according to the invention, for the preparation of a pharmaceutical composition intended for the treatment and/or for the prevention of a disease which is treatable by gene therapy, especially of a genetic disease, of an acquired disease such as cancer or of an infectious disease.
However, such a use is not limited to an application of the somatic gene therapy type. In particular, a vector according to the invention may be used for other purposes such as the production, by the recombinant route in prokaryotic or eukaryotic cells, of product(s) of expression encoded by at least one of the genes of interest. For example, it is possible to envisage the coexpression of two genes of interest in a dicistronic expression vector using a nucleotide sequence according to the invention. The coexpression of a gene for resistance to an antibiotic as a second cistron may make it possible to increase the expression of a first cistron. It is possible to obtain a mature product by coexpression of two genes for which the product of expression of one allows the maturation of the polypeptide encoded by the other (for example polypeptide precursor and a protease cleaving the precursor into a mature polypeptide). In this case, it is possible to use prokaryotic cells (E. coli and the like), lower eukaryotic cells (yeast, fungus, insect and the like) or animal cells. Said product of expression of interest will then have to be harvested and optionally purified from the supernatant or from the cell culture by conventional techniques. Another possible use consists in the production of transgenic animals which have integrated into their genome a cassette for the expression of one or more genes of interest and comprising a nucleotide sequence according to the invention. These may be mice, rats, rabbits, fish, primates or farm animals (bovines, ovines, porcines and the like). The techniques for generating these transgenic animals are known. The polypeptide of interest may be recovered in a conventional manner, for example, from the biological fluids (blood, milk and the like) of the animal.
The invention also relates to a pharmaceutical composition comprising, as therapeutic or prophylactic agent, a vector, a viral particle or a cell according to the invention or a polypeptide of interest obtained in accordance with the use according to the invention, in combination with a pharmaceutically acceptable vehicle.
A pharmaceutical composition according to the invention may be manufactured in a conventional manner. In particular, a therapeutically effective quantity of such an agent is combined with a carrier, a diluent or an adjuvant which is acceptable. It may be administered by any route of administration, in a single dose or in a dose repeated after a certain time interval. The intravenous, intramuscular, intrapulmonary (optionally by aerosolization) or intratumor administration will be preferred. The quantity to be administered will be chosen according to various criteria, in particular the use as a treatment or as a vaccine, the route of administration, the patient, the type of disease to be treated and its state of progression, the duration of the treatment, the vector selected and the like. As a guide, a pharmaceutical composition according to the invention comprises between 104 and 1014 pfu (plaque forming unit), advantageously between 105 and 1013 pfu and, preferably, between 106 and 1011 pfu of viral particles. A vector-based composition may be formulated in the form of doses comprising from 0.01 to 100 mg of DNA, preferably from 0.05 to 10 mg and most preferably from 0.1 to 5 mg.
The formulation may also include, alone or in combination, a diluent, an adjuvant or an excipient which is pharmaceutically acceptable, as well as a solubilizing, stabilizing or preserving agent. The composition may be presented in a single dose or in multidoses in liquid form or in dry form (freeze-dried product and the like) which can be reconstituted immediately before use with an appropriate diluent.
Moreover, the invention relates to a method of treating genetic diseases, cancers and infectious diseases according to which a therapeutically effective quantity of a vector, of a viral particle or of a cell according to the invention is administered to a patient requiring such a treatment. According to a first therapeutic protocol, they can be administered directly in vivo, for example by intravenous injection, intramuscular injection, intratumor injection or by aerosolization into the lungs. Alternatively, it is possible to adopt an ex vivo gene therapy protocol which consists in collecting the cells from a patient (bone marrow stem cells, peripheral blood lymphocytes and the like), in transfecting them with a vector according to the invention and in culturing them in vitro before reimplanting them into the patient.
Finally, the invention relates to the use of a vector, of a viral particle or of a pharmaceutical composition according to the invention for the transfection or infection of pluripotent cells, especially pluripotent cells of the central nervous system.